Properties of microfiltration membranes: the effects of adsorption and shear on the recovery of an enzyme

An experimental study of the interaction of the enzyme yeast alcohol dehydrogenase (YADH) with microfiltration membranes has been carried out. Most measurements were made with capillary pore inorganic membranes (Anopore) with some comparative measurements being made with polymeric membranes of low protein affinity (Durapore). It has been shown that the prolonged exposure of the enzyme to the inorganic membrane under low-shear conditions (slow recycle) resulted in a loss of enzyme activity. Under filtration conditions, the membrane permeation rate decreased continuously with time. This decrease could be quantified using the standard blocking filtration law, which describes a decrease in pore volume due to deposition of enzyme on the walls of the pore. No significant loss in activity of permeating enzyme occurred under solution conditions where the enzyme was stable. However, a significant loss of such activity occurred under solution conditions where the enzyme was slightly unstable. The experiments indicate that the likely mechanism for activity loss is a membrane/enzyme interaction resulting from a shear induced deformation of the enzyme structure. Two conclusions of practical importance are drawn from the work. (c) 1992 John Wiley & Sons, Inc.     

Properties of microfiltration membranes: flux loss during constant pressure permeation of bovine serum albumin

An experimental study of permeation of dilute BSA solutions (filtration) at microfiltration membranes has been carried out. Most measurements were made with capillary pore aluminum oxide membranes, with some comparative measurements with tortuous and capillary pore polymeric membranes. In all cases, a continuous and substantial decrease in the rate of permeation with time was observed. This decrease in permeation with time was observed. This decrease in permeation rate was due neither to concentration polarization nor to protein adsorption alone. However, it could be quantified using the standard blocking filtration law, which describes a decrease in pore volume due to deposition of protein on the walls of the pore. The maximum calculated thickness of the deposited layers was 55nm on the walls of 200-nm diameter pores. This phenomenon is quite different to adsorption of protein at such surfaces, this latter giving only sub-monolayer or monolayer protein coverage under the conditions studied.     

Influence of fermentation conditions and microfiltration processes on membrane fouling during recovery of glucuronane polysaccharides from fermentation broths.

We have investigated the recovery of exopolysaccharides produced by Sinorhizobium meliloti M5N1 CS bacteria from fermentation broths using different membrane filtration processes: cross-flow filtration with a 7 mm i.d. tubular ceramic membrane of 0.5-microm pores under fixed transmembrane pressure or fixed permeate flux and dynamic filtration with a 0.2 microm nylon membrane using a 16-cm rotating disc filter. With the tubular membrane, the polysaccharide mass flux was mainly limited by polymer transmission that decayed to 10% after 90 min. The mass flux of polymer produced under standard fermentation conditions (70 h at 30 degrees C) stabilized after 70 min to 15 g/h/m(2). This mass flux rises to 36 g/h/m(2) when the mean stirring speed during fermentation is increased and to 123 g/h/m(2) when fermentation is extended to 120 h. In both cases, the mean molecular weight of polysaccharides drops from 4.0 10(5) g/mol under standard conditions to 2.7 10(5) g/mol. A similar reduction in molecular weight was observed when the fermentation temperature was raised to 36 degrees C without benefit to the mass flux. These changes in fermentation conditions have little effect on stabilized permeate flux, but raise significantly the sieving coefficient, due probably to molecular weight reduction and the filamentous aspect of the polymer as observed from SEM photographs. The polymer-mass flux was also increased by reducing transmembrane pressure (TMP) and raising the shear rate by inserting a rod in the membrane lumen. Operation under fixed permeate flux instead of constant TMP inhibited fouling during the first 4 h, resulting in higher sieving coefficients and polymer mass fluxes. The most interesting results were obtained with dynamic filtration because it allows operation at high-shear rates and low TMP. Sieving coefficients remained between 90 and 100%. With a smooth disc, the polysaccharide mass flux remained close to 180 g/h/m(2) at 1500 rpm and cell concentrations from 1 to 3 g/L. When radial rods were glued to the disc to increase wall shear stress and turbulence, the mass flux rose to 275 g/h/m(2) at the same speed and cell concentration.     

Membrane fouling by cell-protein mixtures: in situ characterisation using multi-photon microscopy

Fouling of the membrane by cell and protein mixtures can result in severe flux declines, leading to the eventual need to clean or replace the membrane. In this study multi-photon microscopy, a fluorescence-based technique is used to 3-D image in situ the fouling of microfiltration membranes by suspensions containing combinations of washed yeast, bovine serum albumin (BSA) and ovalbumin. Appropriate fluorescent labelling allows the three foulant species to be clearly identified. Images correlate well with filtration data and clearly show the cake of yeast cells capturing protein aggregates. The proteins exhibited very different filtration behaviour. When filtering washed yeast together with ovalbumin and/or a 50:50 mixture by mass of BSA and ovalbumin, the ovalbumin fouling dominates the system. Capture of aggregates by the cake did not reduce fouling of the membrane by the protein and increased the resistance of the cake. For mixtures of BSA and washed yeast, the presence of a cake of yeast cells did reduce fouling of the membrane by the protein, however, the extra resistance due to the cake resulted in a flux lower than that when filtering BSA alone.     

Piezoelectric tuning fork probe for atomic force microscopy imaging and specific recognition force spectroscopy of an enzyme and its ligand

Piezoelectric quartz tuning fork has drawn the attention of many researchers for the development of new atomic force microscopy (AFM) self‐sensing probes. However, only few works have been done for soft biological materials imaging in air or aqueous conditions. The aim of this work was to demonstrate the efficiency of the AFM tuning fork probe to perform high‐resolution imaging of proteins and to study the specific interaction between a ligand and its receptor in aqueous media. Thus, a new kind of self‐sensing AFM sensor was introduced to realize imaging and biochemical specific recognition spectroscopy of glucose oxidase enzyme using a new chemical functionalization procedure of the metallic tips based on the electrochemical reduction of diazonium salt. This scanning probe as well as the functionalization strategy proved to be efficient respectively for the topography and force spectroscopy of soft biological materials in buffer conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

原子力显微技术在酶学研究中的应用

酶在生物体的生命活动中占有及其重要的地位,机体功能的和谐统一有赖于酶的作用。原子力显微技术(AFM)作为一门新发展起来的技术,为人们认识酶的结构与功能提供了又一新的窗口。AFM能够在生理条件下对生物样品进行三维成像,在分子水平上实时监测生理生化反应。AFM还能够在皮牛顿精度上测定分子间作用力。目前,AFM已用于单分子酶的化学性质及其作用原理的研究。本简述AFM在酶学中的应用情况。     

Evaluation of membranes for use in on-line cell separation during mammalian cell perfusion processes

In this study two microporous hollow fibre membranes were evaluated for their use as cell retention device in continuous perfusion systems. A chemically modified permanent hydrophillic PTFE membrane and a hydrophilized PP membrane were tested. To investigate the filtration characteristics under process conditions each membrane was tested during a long term perfusion cultivation of a hybridoma cell line. In both cultivations the conditions influencing membrane filtration (e.g. transmembrane flux) were kept constant. Filtration behaviour was investigated by monitoring transmembrane pressure and protein permeability. Transmembrane pressure was measured on-line with an autoclavable piezo-resistive pressure sensor. Protein permeability was determined by quantitative evaluation of unreduced, Coomassie stained SDS-PAGE. The membrane fouling process influences the filtration characteristics of both membranes in a different way. After fermentation the PP membrane was blocked by a thick gel layer located in the big outer pores of the asymmetric membrane structure. The hydraulic resistance was higher but the protein permeability was slightly better than of the PTFE membrane. For this reason the PP membrane should be preferred. On the other hand, transmembrane pressure decreases slower when the PTFE membrane is used, which favours this membrane for long term cultivations, especially when low molecular weight proteins (<30 KD) are produced.Abbreviations PP Polypropylene - PTFE Polytetrafluoroethylene     

Supramolecular assembly of VDAC in native mitochondrial outer membranes

The voltage-dependent anion channel (VDAC) is the most abundant protein in the mitochondrial outer membrane (MOM). Due to its localization, VDAC is involved in a wide range of processes, such as passage of ATP out of mitochondria, and particularly plays a central role in apoptosis. Importantly, the assembly of VDAC provides interaction with a wide range of proteins, some implying oligomerization. However, many questions remain as to the VDAC structure, its supramolecular assembly, packing density, and oligomerization in the MOM is unknown. Here we report the so far highest resolution view of VDAC and its native supramolecular assembly. We have studied yeast MOM by high-resolution atomic force microscopy (AFM) in physiological buffer and found VDAC in two distinct types of membrane domains. We found regions where VDAC was packed at high density (approximately 80%), rendering the membrane a voltage-dependent molecular sieve. In other domains, VDAC has a low surface density (approximately 20%) and the pore assembly ranges from single molecules to groups of up to 20. We assume that these groups are mobile in the lipid bilayer and allow association and dissociation with the large assemblies. VDAC has no preferred oligomeric state and no long-range order was observed in densely packed domains. High-resolution topographs show an eye-shaped VDAC with 3.8 nm x 2.7 nm pore dimensions. Based on the observed VDAC structure and the pair correlation function (PCF) analysis of the domain architectures, we propose a simple model that could explain the phase behavior of VDAC, and illustrates the sensitivity of the molecular organization to conditions in the cell, and the possibility for modulation of its assembly. The implication of VDAC in cytochrome c release from the mitochondria during cell apoptosis has made it a target in cancer research.     

Involvement of β‐adrenergic receptor in synaptic vesicle swelling and implication in neurotransmitter release

Secretory vesicle swelling is required for vesicular discharge during cell secretion. The G_αo‐mediated water channel aquaporin‐6 (AQP‐6) involvement in synaptic vesicle (SV) swelling in neurons has previously been reported. Studies demonstrate that in the presence of guanosine triphosphate (GTP), mastoparan, an amphiphilic tetradecapeptide from wasp venom, activates G_o protein GTPase, and stimulates SV swelling. Stimulation of G proteins is believed to occur via insertion of mastoparan into the phospholipid membrane to form a highly structured α‐helix that resembles the intracellular loops of G protein‐coupled adrenergic receptors. Consequently, the presence of adrenoceptors and the presence of an endogenous β‐adrenergic agonist at the SV membrane is suggested. Immunoblot analysis of SV using β‐adrenergic receptor antibody, and vesicle swelling experiments using β‐adrenergic agonists and antagonists, demonstrate the presence of functional β‐adrenergic receptors at the SV membrane. Since a recent study shows vH⁺‐ATPase to be upstream of AQP‐6 in the pathway leading from G_αo‐mediated swelling of SV, participation of an endogenous β‐adrenergic agonist, in the binding and stimulation of its receptor to initiate the swelling cascade is demonstrated.     

Characterizing molecular interactions in different bacteriorhodopsin assemblies by single-molecule force spectroscopy

Using single-molecule force spectroscopy we characterized inter- and intramolecular interactions stabilizing structural segments of individual bacteriorhodopsin (BR) molecules assembled into trimers and dimers, and monomers. While the assembly of BR did not vary the location of these structural segments, their intrinsic stability could change up to 70% increasing from monomer to dimer to trimer. Since each stable structural segment established one unfolding barrier, we conclude that the locations of unfolding barriers were determined by intramolecular interactions but that their strengths were strongly influenced by intermolecular interactions. Subtracting the unfolding forces of the BR trimer from that of monomer allowed us to calculate the contribution of inter- and intramolecular interactions to the membrane protein stabilization. Statistical analyses showed that the unfolding pathways of differently assembled BR molecules did not differ in their appearance but in their population. This suggests that in our experiments the membrane protein assembly does not necessarily change the location of unfolding barriers within the protein, but certainly their strengths, and thus alters the probability of a protein to choose certain unfolding pathways.     

Electrical excitability of artificial enzyme membranes: II. Electrochemical and enzyme properties of immobilized acetylcholinesterase membranes

This paper deals with aspects of the reciprocal interaction between enzyme activity and the microenvironment or the potential difference in artificial proteinaceous membranes bearing cross-linked acetylcholinesterase. The potential difference resulting from asymmetric substrate injection into the system is recorded as a function of time. The influence of the membrane charge density on both enzyme activity and potential difference is studied by varying the external solution pH. The enzyme specific potential is initiated by local change of pH at the membrane level and the dependence on the buffer strength is studied. The recorded potential difference appears to be the result of the reciprocal interaction between enzyme reaction and the diffusion of substrate or products.     

Immobilization of Candida rugosa lipase on polypropylene microfiltration membrane modified by glycopolymer: hydrolysis of olive oil in biphasic bioreactor

Polypropylene hollow fiber microfiltration membranes (PPHFMM) with improved hydrophilicity and biocompatibility surface were prepared by the plasma-induced graft polymerization of -allyl glucoside and were used to immobilize lipase from Candida rugosa by adsorption. A biphasic enzyme membrane bioreactor (EMR) was assembled with the glycopolymer-modified and enzyme-immobilized PPHFMM. Effect of operating variables on the performance of this biphasic EMR was investigated with the hydrolysis of olive oil. It was found that, at the optimal operational condition, an apparent volumetric reaction rate of about 0.074 mmol/l h can be obtained. This result indicated that the lipase-immobilized PPHFMM exhibited the catalytic efficiency similar to that of some hydrophilic membranes in biphasic EMR, which verified the feasibility of the employment of surface-hydrophilized polypropylene membranes in such EMR.     

EDTA-induced Membrane Fluidization and Destabilization： Biophysical Studies on Artificial Lipid Membranes

The molecular mechanism of ethylenediaminetetraacetic acid （EDTA）-induced membrane destabilization has been studied using a combination of four biophysical techniques on artificial lipid membranes. Data from Langmuir film balance and epifluorescence microscopy revealed the fluidization and expansion effect of EDTA on phase behavior of monolayers of either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine （DPPC） or mixtures of DPPC and metal-chelating lipids, such as N^a,N^a-Bis[carboxymethyl]-N^ε [（dioctadecylamino）succinyl]-L-lysine or 1,2-dioleoyl-sn-glycero-3-[N-（5-amino- 1 -carboxypentyl iminodiacetic acid） succinyl]. A plausible explanation could be drawn from the electrostatic interaction between negatively charged groups of EDTA and the positively charged choline head group of DPPC. Intercalation of EDTA into the lipid membrane induced membrane curvature as elucidated by atomic force microscopy. Growth in size and shape of the membrane protrusion was found to be time-dependent upon exposure to EDTA. Further loss of material from the lipid membrane surface was monitored in real time using a quartz crystal microbalance. This indicates membrane restabilization by exclusion of the protrusions from the surface. Loss of lipid components facilitates membrane instability, leading to membrane permeabilization and lysis.     

Protein composition and biomechanical properties of in vivo-derived basement membranes

Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.     

Role of membrane structure on the filtrate flux during monoclonal antibody filtration through virus retentive membranes

Virus removal filtration is a critical step in the manufacture of monoclonal antibody products, providing a robust size-based removal of both enveloped and non-enveloped viruses. Many monoclonal antibodies show very large reductions in filtrate flux during virus filtration, with the mechanisms governing this behavior and its dependence on the properties of the virus filter and antibody remaining largely unknown. Experiments were performed using the highly asymmetric Viresolve® Pro and the relatively homogeneous Pegasus™ SV4 virus filters using a highly purified monoclonal antibody. The filtrate flux for a 4 g/L antibody solution through the Viresolve® Pro decreased by about 10-fold when the filter was oriented with the skin side down but by more than 1000-fold when the asymmetric filter orientation was reversed and used with the skin side up. The very large flux decline observed with the skin side up could be eliminated by placing a large pore size prefilter directly on top of the virus filter; this improvement in filtrate flux was not seen when the prefilter was used inline or as a batch prefiltration step. The increase in flux due to the prefilter was not related to the removal of large protein aggregates or to an alteration in the extent of concentration polarization. Instead, the prefilter appears to transiently disrupt reversible associations of the antibodies caused by strong intermolecular attractions. These results provide important insights into the role of membrane morphology and antibody properties on the filtrate flux during virus filtration.     

Electrical excitability of artificial enzyme membranes: III. Hysteresis and oscillations observed with immobilized acetylcholinesterase membranes

Experimental evidence for memory and oscillations in artificial acetylcholinesterase membranes is presented. When acetylcholine is injected on one side of an artificial proteinic membrane bearing acetylcholinesterase, a potential difference is recorded as a function of time. The steady-state potential due to the enzyme activity for increasing and decreasing substrate concentrations exhibits a hysteresis loop. The non-linearity of the enzyme reaction coupled with the diffusion constraints cause also some instabilities, such as oscillations of the membrane potential.     

Cellular traction force recovery: An optimal filtering approach in two-dimensional Fourier space

Quantitative estimation of cellular traction has significant physiological and clinical implications. As an inverse problem, traction force recovery is essentially susceptible to noise in the measured displacement data. For traditional procedure of Fourier transform traction cytometry (FTTC), noise amplification is accompanied in the force reconstruction and small tractions cannot be recovered from the displacement field with low signal-noise ratio (SNR). To improve the FTTC process, we develop an optimal filtering scheme to suppress the noise in the force reconstruction procedure. In the framework of the Wiener filtering theory, four filtering parameters are introduced in two-dimensional Fourier space and their analytical expressions are derived in terms of the minimum-mean-squared-error (MMSE) optimization criterion. The optimal filtering approach is validated with simulations and experimental data associated with the adhesion of single cardiac myocyte to elastic substrate. The results indicate that the proposed method can highly enhance SNR of the recovered forces to reveal tiny tractions in cell-substrate interaction.     

Force spectroscopy reveals the presence of structurally modified dimers in transthyretin amyloid annular oligomers

Toxicity in amyloidogenic protein misfolding disorders is thought to involve intermediate states of aggregation associated with the formation of amyloid fibrils. Despite their relevance, the heterogeneity and transience of these oligomers have placed great barriers in our understanding of their structural properties. Among amyloid intermediates, annular oligomers or annular protofibrils have raised considerable interest because they may contribute to a mechanism of cellular toxicity via membrane permeation. Here we investigated, by using AFM force spectroscopy, the structural detail of amyloid annular oligomers from transthyretin (TTR), a protein involved in systemic and neurodegenerative amyloidogenic disorders. Manipulation was performed in situ , in the absence of molecular handles and using persistence length‐fit values to select relevant curves. Force curves reveal the presence of dimers in TTR annular oligomers that unfold via a series of structural intermediates. This is in contrast with the manipulation of native TTR that was more often manipulated over length scales compatible with a TTR monomer and without unfolding intermediates. Imaging and force spectroscopy data suggest that dimers are formed by the assembly of monomers in a head‐to‐head orientation with a nonnative interface along their β‐strands. Furthermore, these dimers stack through nonnative contacts that may enhance the stability of the misfolded structure.     

Measurement of the size and structure of natural aquatic colloids in an urbanised watershed by atomic force microscopy

Atomic force microscopy (AFM) in tapping mode was used to determine the conformation of humic substances and aquatic colloids from rivers in an urban catchment in the West Midlands, U.K. Humic macromolecules were shown to have a size of about 1–3 nm in agreement with the literature, indicating that the preparation methods and the AFM were both performing satisfactorily. Three types of natural aquatic colloids were observed by AFM. Firstly, a surface coating about 1–5 nm thick, likely composed of organic and oxide material flattened by drying and interaction with the AFM tip. Secondly, small irregular, globular material between 1 and 70 nm in size, again most likely made of oxide and organic material. Lastly, fibrillar material was present which was 1–10 nm in diameter and 10–1000 nm in length. Most likely this material was microbially produced (muco-) polysaccharides. Size distributions of colloids from all samples, regardless of sample site and sample preparation, indicated colloids with a fairly low polydispersity and with particle numbers dominated by material <10 nm.     

Fluorescence correlation spectroscopy in the nanosecond time range: Photon antibunching in dye fluorescence

Possibilities for the use of fluorescence correlation spectroscopy in the nanosecond time range are demonstrated. The experiment is based on a cw argon ion laser, a microfluorimeter, two photon detectors, and a time-to-analog converting system. Experiments using solutions of rhodamine 6G and pyronine G in water at concentrations of about 20 molecules per sample volume are reported. The photon anticorrelation component decaying with a time constant close to the excited state lifetime was observed.